Process for the production of antibiotics

ABSTRACT

A process for producing Rifamycin B substantially free from analogues sub-products and with high productivity. According to this process a mutant of the microorganism of the species Streptomyces Albovinaceus is cultivated in an aqueous nutrient medium of controlled initial pH containing assimilable sources of carbon, nitrogen and inorganic salts, in submerged aerobic conditions: Rifamycin B is produced which is then recovered by conventional methods.

Elite Sttes atent 1 Pasqualucci et al.

[ Mar. 18, 1975 PROCESS FOR THE PRODUCTION OF ANTIBIOTICS Inventors: Carmine Renato Pasqualucci, Milan;

Giuseppe Zucco, Rovereto, both of Italy Assignee: Arckifar lndustrie Chimiche Del Trentino S.p.A., Rovereto, Trento, Italy Filed: Oct. 12, 1973 Appl. No.: 405,829

Foreign Application Priority Data Nov. 29. 1972 Italy 32248/72 U.S. Cl 195/80 R Int. Cl Cl2d 9/00 Field of Search 195/80 R [56] References Cited UNlTED STATES PATENTS 2,999,048 9/196] Donovick et al 195/80 R 3,150,046 9/1964 Sensi et a] i. 195/80 R Prinmry Examiner-Lionel M. Shapiro Assistant Examiner-Robert J. Warden [57] ABSTRACT 1 Claim, N0 Drawings PROCESS FOR THE PRODUCTION OF ANTIBIOTICS This invention relates to production of rifamycin B by fermentation of the mutant called Streptomyces GS/A.

The new mutant high producer of rifamycin B, as isolated by the applicant, has been obtained by repeated mutagenic treatments with UV. radiations and subsequent selections of colonies exhibiting an improved production of rifamycin B, from a culture specimen of the Streptomyces albovinaceous stock ATCC 12,951 and which is the subject of US Pat. No. 2,999,048.

It is known that the nancimycin described in US. Pat. No. 2,999,048 is in practice an antibiotic called by others rifamycin B (see French Pat. No. 2,043,847) and this antibiotic can be produced by submerged aerobic fermentation in a nutrient medium containing a source of assimilable carbon, assimilable nitrogen and mineral salts agitated at a controlled temperature of the microorganism ATCC 12951 as described in the abovementioned US. patent. The productivity of this microorganism is not, however, such as to allow exploitation in conditions which would be economically attractive from the industrial viewpoint.

We have now, surprisingly, found that a mutant of the microorganism known as ATCC 12,951 is extremely activein producing rifamycin B. This mutant has been called by the applicant Streptomyces G5/A and has been deposited on May 23, 1972 with the public collector CENTRAALBUREAU VOOR SCHIM- MELCULTURES Baarn Nederland, where it is identified by the number 6/5 A.

Furthermore the Rifamycin B obtained by this invention does not contain substantial quantities of secondary analogous products. This is also a significant advantage as far as the effects of further purification of the final product are concerned.

This invention relates to an improved process for producing Rifamycin B substantially free from analogues sub-products and with high productivity. According to this process a mutant of the microorganism of the species Streptomyces Albovinaceus is cultivated in an aqueus nutrient medium of controlled initial pH containing assimilable sources of carbon, nitrogen and inorganic salts, in submerged aerobic conditions: Rifamycin B is produced which is then recovered by conventional methods.

The streptomyces G5/A of this invention presents taxonomic characteristics different from those of other rifamycin-producing organisms as shown in the following tables.

For taxonomic studies, as also for some additional media, the methods and culture media described by Shirling and Gottlieb (Int. system Bact., 16 313340, 1966) have been used.

For colour description the colour tables of H. Prauser have been used (Prauser H. 1964. Aptness and applica tion of colour codes for exact description of colours of streptomycetes. Zeitschrift f.Al1g. Mikrobiologie, 4, 9598).

From comparison of the data obtained it is obvious that the streptomyces G5/A differs from other strains in various and fundamental characteristics.

Table 1 sets out the general characteristics of the cul- 3O tures of the strain of this invention and of the tradi- TABLE 1 GENERAL CHARACTERISTICS OF THE CULTURES Str. Str. Str. Str. GS/A Albovinaceus Mediterranei Mediterranei 4107/A. B 2847 ATCC 12951 ATCC 21271 ATCC 13685 Spores Absent Ellipsoidal Absent Rod-like Cylindric, with Ellipsoidal, with or oval a smooth surface a smooth surface Aerial Absent White to violet Absent Mealy, pinkish- Pink to white, White with simple mycelium sporophores with white, sporophores sporophores straight branches spores in chains, with spores in spores in chains no spirals chains, no spirals Vegetative Dark brown Light pink to Yellow-orange Yellowish to Red to orange Colourless to mycelium to light brown to strong dark pink-orange yellowish brown with yellow tinges of orange to dark orange Soluble Dark to light Mostly absent; Yellowish green Absent or Brown to purple Yellow orange to pigments brown to yellow when there are to strong yellow yellowish yellow brown with a green any they present brown tinge a dark red colouration Str. (IS/A mutant subject of this invention:

Slr. Allmvinuccm 11.8. pntcnt 2.999.041;

Str. Mvtlltvnnuci /\'l('( 21271 Swim pntcnl 477551; Str. Mctlilt-rrnnci Al'((' 1.1085 1|.S. pntcnt LUNHAW); 4107M, Journnl Antibiotic XVll 2! 1904;

1121147 Journal Antibiotic XXIV, 1110. I971.

TABLE 2 ASSlMlLATlON OF CARBON COMPOUNDS Carbon source Str. GS/A Str. Albovinaceus Str. Mediterranei Str. Mediterranei 4l07 A B 2847 ATCC l295l ATCC 2l27l ATCC 13685 Glucose H H H H H H Arabinose H H Hl- H Rhamnose H H H +t+ Xylose H H 4-ii Galactose H H H +H Fructose H H -H+ H H Mannose i H H H H H Lactose i' H i H t Maltose i H ll- Sucrose H H H H Raffinose i H H Dextrin H H Not det Not dett lnulin i i H Dulcitol H Glycerine H H H 4-H- Not det. H Mannitol H H lnositol i H -H- Sorhitol Na-suceinate Not det. Na-acetate (jlieolia Not det. Not deti Not det. Saliciii Not det. Starch (soluble) Cellulose Not dct. Not det. Not det. Na-eitrate -lll- Excellent utilization -l+ Very positive utilization Positive utilization i Doubtful utilization Negative utilization l Sec. Sensi. P., Thiemann .l. E. Progress in lndustrial Microbiology, Vol. 6, 2l-60, 1967(DJ. Hockenhull, Ed.) London Heywood Books.

London.

TABLE 3 BIOCHEMICAL CHARACTERISTICS Str4 Str. Stri Str. (iS/A Alhovinaeeus Mediterraiiei Mediterranei 4l()7 A ll 2X47 ATCC I295] ATCC 2l27l ATCC U685 Agarized Slight Not determined Not digested Slight digestion Not determined Not determined Czi-mzilate digestion Agarized Not hydrolysed Not determined Not hydrolysed Not hydrolysed Not determined Not determined t 'rosine lelatine No liquefaction, Liquefaction No liquefaction Slight to no Strong lique- Rapid liquefaction no pigment liquefaction faction yellowish brown no pigment brown pigments Nitrates Not reduced Reduced Not reduced Not reduced Rapid liquefaction.

yellowish brown pigments Litmus No peptonisation No variation No peptonisation No peptonisation Peptonisution Low peptonisation milk or coagulation or coagulation or Coagulation. without coagulation slight alkaline reaction Medium (1 Formation of Not determined Formation of Formation of H S Not determined Not determined of Sliirling H S; no H S; no no pigmentation St (jottlieh pigmentation pigmentation (Ph acetate indicator) Dextrin Hydrolysed Positive Not hydrolysed Positive Not determined Not determined hydrolysation hydrolysis Starch I'LYtII'ULYSLd tosilive Not hydrolysed Positive No hydrolysis Not determined (soluble) l \tll'()l VSilllUll hydrolysis l i Not \l\'l\'lllllll\\i Illtdlh that the lllt'tlllllll in t iieslioii has not been used In detei'iiiiiic the biochemical cliai'actcrislit'sv lloiveu-i, as may he seen in the following taliles.

it is used to ileleiiiiiiic iilhci cliiiizicleilslics TABLE 4 CHARACTERISTICS OF THE CULTURES OF SOME STANDARD MEDIA Str.

Str. Str. Media Str. GS/A Alhovinaceus Mediterranei Mcditcrranei 4l07 A: B 2847 ATCC l295l ATCC 21271 ATCC 13685 Nutrient Agar G-Ciood. high, Ggood, surface G-Moderate, G-Moderate, (i-Moderate G-Poor to (after Waksman) deeply wrinkled rough, hard and flat, surface surface smooth VM-Yellow moderate surface crusty slightly rough VM-Melon to orange colourless VM-Greyish VM-Light brown VM-Light brown orange AM-Mealy, AM Poor, white (Oc4s) with orange with slight tinge AM-Pinkish white to pale greyish AM-Absent tinge (Oc3r) oforange white SP-None (Rdg Ll |5""'-f) SP-Dark brown AM-Pale Pink AM-Absent SP-Absent R-Colourless to with reddish (Oc7a) SP-Absent pale yellow orange tinge OcSr) SP-Light yellow- SP-None ish brown (Coo2a) to pale pinkish brown TABLE 4 :Qontinasi.

CHARACTERISTICS OF THE CULTURES OF SOME STANDARD MEDIA Str. Str. Str. Media Str. GS/A Albovinaceus Mediterranei Mediterranei 4l07 A. B 2847 ATCC l 295 l ATCC 2 l 271 ATCC l3685 Bennett's Agar G-Good, high, G-Good, rough, G-Good, high, G-Good G-(jood (after Waksman) very wrinkled surface high, hard surface very VM-Yellowish VM-Dark orange surface and crusty rough tending to to yellowish Not described VM-Dark brown VM-Strong red VM-Strong red or orange yellow orange or vice with tinges of (Or3r) brown (O4r) AM-Pink versa light red to AM-White-cream AM-Absent SP-Light amber AMMealy, greyish brown with light pink SP-Light brown abundant, pale (O5r-Oc4s) tinge (Coo7a) with tinges or pink AM-Absent SP-Strong red orange SP-Brown. SP-Dark brown (Or4r) (Oc3r) residues with tinges ofreddish to a slight greenish brown (Oc5r-Coo4r) Medium 2 of G-Good, high, G-Good, flat, G-Good, high, GAbundant, (J-Good, rough G-Abundant Shirling & very wrinkled surface regular, surface very coarse VM-Reddish rolled up Gottlieb afligvglgte l hard rough VM-Colourless brown to orange AM-White at surface VM-Red with VM-Strong to yellowish AM-Mealy, fine the edge VM-Brown with orange tinge orange (Oc2r) AM-Whitish pale orange R-Colourless tings gflavender, (Or3r) AM-Absent SP-lntense amber SPBrown SP- to orange AMV Vhite (Wl); SP-Strong red to (Oc5s-Oc3r) mealy brown (O4r) AM-Absent SP-Strong red SP Dark brown (Or4r) with light red tinge Medium 3 of G-Moderate to G-Good, high, (Iv-Moderate, flat G-Moderate G-White Orange Shirling & good, flat, with surfacerougll. VM-Yellow surface smooth to light orange Gottlieb high edges hard and crusty brown (Coo2r) VM-Crystalline with a rough Not described VM-Light brown VM-Pale. with AM-Residues. to yellowish surface with orange tinge light yellow white at the edge AM-Whitish AM-Mealy. (centre of the tinge (Co6a) ofthe colony (Wl) with pink tinge abundant pinkish colony) to orange AM-White (W l SP-Yellow brown SP-Weak, white with red tinges mealy (Qgolr) yellowish SP-None (edges ofthe SP-Colourless to colony (Oc3r-O3r) pale pink (OrZa) AM-Absent SP-Ash-grey with slight tinge of brown (C0o6t) Medium 4 of G-Moderate, G-Moclerate to G-Good, flat G-Sparse G-White, yellow G-Moderate Shirling & slightly elevated, good surface smooth VM-Colourless to orange colourless Gottlieb surface wrinkled VM-Pale with a VM-Salmon pink light orange red AM-Abundant, AM-Poor, white VM-Slightly slight tinge ofgrey (03a) AM-Sparse, mealy pale orange at the edges brown with orange AM-White (W1) AM-Residues, white SP-Orange, R-Colourless tinge to intense SP-Absent white (W1) SP-Absent residues SP-None orange SP-Pale orange (Oc3r-Oc-2r) (002a) AM-Absent SPLight brown lavender (OcSm) Medium 5 of G-Moderate. flat, G-Good, high, G-Good, flat G-Moderate, Shirling & lightly wrinkled hard rough rough surface surface smooth Gottlieb VM-Pale orange surface VM-lntense VM-Light pinkish Not described Not described (OcZa) VM-Light orange orange (OZr) orange AMAbsent to brown (Oc2a to AM-Absent AMAbsent SP-Absent Oc4r) SP-Light yellow SPLight yellow AM-Sparse, pale (C020) pink with brown tinges (005a) Medium 6 of G-Moderate, flat G-Moderate, flat, G-Moderate, flat G-Moderate, flat Shirling & smooth surface smooth surface smooth surface surface slightly Gottlieb VM-Light brown VM-Pale (C0521) VM-Pale (CoSa) rough to pale (CooSb) AM-Very sparse, AM-Absent VM-Light brown Not described Not described AM-Absent white (Wl) SP-Absent yellow tinge SP-Yellow with a SP-Light, pale (C0o4a) slight green brown (Coo3m) t n eg mr H 7 a. a 7 Medium 7 of G-Moderate, G-Good, high, G-Good, high G-Moderatc, G-Moderate, Shirling & flat surface hard rou h VM-Not dark colourless Gottlieb rough rough su ace described front 2 VM-Light brown surface AM-Not described yellow to yellow orange (Oc2r) VM-Light brown VM-lntcnse SP-Not described oran e AM-Poor, white AM-Abscnt with orange orangetO'lr) AM- eak, mealy RColourlcss SP-Ycllow with to brown AM-Ahscnt white to SP-None a slight tinge tinges (Oc3r SP-Light pinkish white olgrccn (Cooflm) to Oc4r) orange (OcZa) AM-Sparsc; SP-Nonc light pink with brown tinge (Oc5a) SP-Reddish brown (O4r) CHARACTERISTICS OF THE CULTURES OF SOME STANDARD MEDIA After 48 hours of growth on a reciprocating shaker at 28C. (v/v) of the vegetative growth is transferred to 500 ml. Erlenmeyer flask fitted with a baffle and containing 100 ml. of the following medium:

The inoculated flasks were incubated at 28C. on a rotary shaker at 250 rpm. with a 5 cm. stroke for 148-1- 56 hours. The yield of rifamycin B was from 4000 4500 mcg/ml.

EXAMPLE 2 The microorganism known as G5/A is grown in the same conditions described in Example 1 for the vegetative phase.

The following fermentation medium has used in the final phase.

Str. Str. Str. Media Str. GS/A Albovinaceus Mediterranei Mediterranei 4l07 A B 2847 ATCC 12951 ATCC 2l27l ATCC I3685 Agarizcd G-Good, sur- G-Good. flat, G-Moderatc, G-Regular G-Good'. red GModcrate Ca-malatc face slightly surface smooth flat, surface VM-Colourless to black pale yellow elevated and VM-Yellowish slightly AM-Whitish AM-Mealy. redorange, penc wrinkled (C0221) rough with a pink dish orange [rating into VM-Faded AM-White (Wl) VM-Pale orange tinge in part the medium orange to yellow SP-Colourless (Oc2a) SP-Absent light yellow AM-Fine. white brown (Oc2a to to li ht yellow AMAbsent orange R-Pale from Oc3r) (Co a) SP-Yellow SP-Purple, yellow orange AMAbsent brown (Coo2r) traces to Chamois SP-Brown red to ochre yellowish brown (Rdg.XV l5-()) (Or 2a Coo2a) SP-Chamois (Rdg.

XXX l9"-6l G Growth AM aerial mycclium R reverse VM vegetative mycelium SI7 soluhlt: pigmclfl Rdg R. Ridgway Color standards and color nomenclature. Washington. l9l2 The following examples illustrates the invention but r are not limitative. Peanut meal 25 25 (Sjoybean meal g. 5

. ucose EXAMPLE I Propylene glycol 5 Glycerol g. 40 A culture of Streptomycen G5/A 1s inoculated into a Hlslaivy ggc u g. .3 500 ml. Erlenmeyer flask containing ml. of mek 4 5: dium composed as follows: 8 +7 2 (I35 Ammonium molybdate' g. 0.00] 30 Mnso..4H.o g. 0.004 CoCl .6l-ll?O g. 0.002 Meat extract 8- 5 @3 i g extract s 5 Fesol'imo 5: 0101 FP Q g- 5 Water to l,000 ml. ilSClll hydrolysate g. I 50 pH (adjusted with buffer) 6.6 6.7 after sterilization.

ucose g. N 10 g. 15 35 ai water to L000 Fermentation was conducted as in Example 1. The

maximum strength of rifamycin B reached was 5400 mcg/ml. In chromatography no appreciable quantities of products similar to rifamycin were found.

EXAMPLE 3 The microorganism known as G5/A was grown in the same conditions described in Example 1, using however 2,000 ml. flasks containing 500 ml. of medium; after 48 hours 3 flasks were used to inoculate 90 litres of the same medium in a stainless steel fermentor.

Vegetative growth took place with agitation (500 r.p.m;) and aeration (l v/v/m) at 28C.

After 36 hours about 7 percent in volume of mycelial growth was obtained: this second vegetative phase was used to inoculate a 1,300 litre stainless steel fermentor containing 900 litres of fermentation medium, as described in Example 2.

Growth of the organism took place at 28C with agitation (170 rpm.) and aeration (l v/v/min) and overpressure of 0.2 kg/cm After hours of fermentation the spectrophotometric titre was 5200/5500 rnl. Chromatographic analysis showed only rifamycin B to be substantially present.

What we claim is:

1. A process for producing Rifamycin B, comprising:

cultivating the mutant known as Streptomyces G 5/A capable of high productivity, in a buffered nutrient medium containing assimilable sources of carbon. nitrogen and inorganic salts under submerged aero- 

1. A PROCESS FOR PRODUCING RIFAMYCIN B, COMPRISING: CULTIVATING THE MUTANT KNOWN AS STREPTOMYCES G 5/A CAPABLE OF HIGH PRODUCTIVITY, IN A BUFFERED NUTRIENT MEDIUM CONTAINING ASSIMIABLE SOURCES OF CARBON, NITROGEN AND INORGANIC SALTS UNDER SUBMERGED AEROBIC CONDITIONS. 